Separation matrix

1. A separation matrix comprising Fc-binding ligands covalently coupled to a porous support having a ligand content of at least 11 mg/ml, wherein:

a) said ligands comprise multimers of alkali-stabilized Protein A domains,

b) said porous support comprises cross-linked polymer particles having a volume-weighted median diameter (d50, v) of 56-70 micrometers, a dry solids weight of 55-80 mg/ml, and a pore size corresponding to an inverse gel filtration chromatography Kd value of 0.69-0.85 for dextran of Mw 110 kDa,

wherein the stability of the separation matrix increases with increasing ligand content, and

wherein said alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by, or having at least 80% identity to, SEQ ID NO: 53,

(SEQ ID NO: 53)

X 1 Q X 2 AFYEILX 3 LP NLTEEQRX 4 X 5 F IX 6 X 7 LKDX 8 PSX 9 S

X 10 X 11 X 12 LAEAKX 13 X 14 NX 15 AQ

wherein individually of each other:

X 1 =A, Q, or is deleted

X 2 =E, K, Y, T, F, L, W, I, M, V, A, H or R

X 3 =H or K

X 4 =A or N

X 5 =A, G, S, Y, Q, T, N, F, L, W, I, M, V, D, E, H, R or K

X 6 =Q or E

X 7 =S or K

X 8 =E or D

X 9 =Q, V, or is deleted

X 10 =K, R, A, or is deleted

X 11 =A, E, N, or is deleted

X 12 =I or L

X 13 =K or R

X 14 =L or Y

X 15 =D, F, Y, W, K or R.

2. The separation matrix of claim 1 , wherein said cross-linked polymer particles comprise cross-linked polysaccharide particles.

3. The separation matrix of claim 1 , wherein said cross-linked polymer particles comprise cross-linked agarose particles.

4. The separation matrix of claim 1 , wherein said multimers comprise tetramers, pentamers, hexamers or heptamers of alkali-stabilized Protein A domains.

5. The separation matrix of claim 1 , wherein said multimers comprise hexamers of alkali-stabilized Protein A domains.

6. The separation matrix of claim 1 , having a 10% breakthrough dynamic binding capacity for IgG of at least 45 mg/ml at 2.4 min residence time or a 10% breakthrough dynamic binding capacity for IgG of at least 60 mg/ml at 6 min residence time.

7. The separation matrix of claim 6 , wherein the10% breakthrough dynamic binding capacity for IgG at 2.4 min or 6 min residence time is reduced by less than 20% after incubation 31h in 1.0 M aqueous NaOH at 22+/−2 C.

8. The separation matrix of claim 1 , having a dissociation constant for IgG2 of below 0.2 mg/ml in 20 mM phosphate buffer, 180 mM NaCl, pH 7.5.

9. The separation matrix of claim 1 , wherein said alkali-stabilized Protein A domain has a sequence defined by SEQ ID NO: 11 without linker region amino acids 1-8 and 56-58.

10. The separation matrix of claim 1 , wherein said alkali-stabilized Protein A has a sequence defined by SEQ ID NO:4-7, without linker region amino acids 1-8 and 56-58 wherein the amino acid residue at the position corresponding to position 11 in SEQ ID NO:4-7 is, or has been mutated to, a glutamic acid or a lysine.

11. The separation matrix of claim 1 , wherein said alkali-stabilized Protein A has a sequence defined by SEQ ID NO:4-7, without linker region amino acids 1-8 and 56-58 wherein the amino acid residue at the position corresponding to position 40 in SEQ ID NO:4-7 is, or has been mutated to, a valine.

12. The separation matrix of claim 1 , wherein individually of each other:

X 1 =A or is deleted, X 2 =E, X 3 =H, X 4 =N, X 6 =Q, X 7 =S, X 8 =D, X 9 =V or is deleted, X 10 =K or is deleted, X 11 =A or is deleted, X 12 =I, X 13 =K, X 14 =L.

13. A method of isolating an immunoglobulin, comprising the steps of:

a) contacting a liquid sample comprising an immunoglobulin with a separation matrix according to claim 1 ,

b) washing said separation matrix with a washing liquid,

c) eluting the immunoglobulin from the separation matrix with an elution liquid, and

d) cleaning the separation matrix with a cleaning liquid.

14. The method of claim 13 , wherein the cleaning liquid comprises 0.1-1.0 M NaOH or KOH.

15. The method of claim 14 , wherein steps a)-d) are repeated at least 10 times.

16. A separation matrix comprising Fc-binding ligands covalently coupled to a porous support, having ligand content of at least 15 mg/mL and a pore size corresponding to an inverse gel filtration chromatography Kd value of 0.69-0.85 for dextran of Mw 110 kDa,

wherein the stability of the separation matrix increases with increasing ligand content,

wherein said ligands comprise multimers of alkali-stabilized Protein A domains, and

wherein said alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by, or having at least 80% identity to, SEQ ID NO: 53,

(SEQ ID NO: 53)

X 1 Q X 2 AFYEILX 3 LP NLTEEQRX 4 X 5 F IX 6 X 7 LKDX 8 PSX 9 S

X 10 X 11 X 12 LAEAKX 13 X 14 NX 15 AQ

wherein individually of each other:

X 1 =A, Q, or is deleted

X 2 =E, K, Y, T, F, L, W, I, M, V, A, H or R

X 3 =H or K

X 4 =A or N

X 5 =A, G, S, Y, Q, T, N, F, L, W, I, M, V, D, E, H, R or K

X 6 =Q or E

X 7 =S or K

X 8 =E or D

X 9 =Q, V, or is deleted

X 10 =K, R, A, or is deleted

X 11 =A, E, N, or is deleted

X 12 =I or L

X 13 =K or R

X 14 =L or Y

X 15 =D, F, Y, W, K or R.

17. The separation matrix of claim 16 , having a sequence defined by SEQ ID NO:33 made up of alkali-stabilized Protein A domain defined by SEQ ID NO:11, without linker region amino acids 1-8 and 56-58.

18. The separation matrix of claim 16 , wherein said cross-linked polymer particles comprise cross-linked agarose particles.

19. The separation matrix of claim 16 , having a 10% breakthrough dynamic binding capacity for IgG of at least 45 mg/ml at 2.4 min residence time or a 10% breakthrough dynamic binding capacity for IgG of at least 60 mg/ml at 6 min residence time.

20. The separation matrix of claim 19 , wherein the 10% breakthrough dynamic binding capacity for IgG at 2.4 min or 6 min residence time is reduced by less than 20% after incubation 31h in 1.0 M aqueous NaOH at 22+/−2 C.

21. The separation matrix of claim 16 , wherein said multimers comprise tetramers, pentamers, hexamers or heptamers of alkali-stabilized Protein A domains.

22. The separation matrix of claim 16 , having a dissociation constant for IgG2 of below 0.2 mg/ml in 20 mM phosphate buffer, 180 mM NaCl, pH 7.5.

23. The separation matrix of claim 16 , wherein individually of each other:

X 1 =A or is deleted, X 2 =E, X 3 =H, X 4 =N, X 6 =Q, X 7 =S, X 8 =D, X 9 =V or is deleted, X 10 =K or is deleted, X 11 =A or is deleted, X 12 =I, X 13 =K, X 14 =L.

24. A method of isolating an immunoglobulin, comprising the steps of:

a) contacting a liquid sample comprising an immunoglobulin with a separation matrix according to claim 16 ,

b) washing said separation matrix with a washing liquid,

c) eluting the immunoglobulin from the separation matrix with an elution liquid, and

d) cleaning the separation matrix with a cleaning liquid,

wherein the cleaning liquid comprises 0.1-1.0 M NaOH or KOH.

25. The method of claim 24 , wherein steps a)-d) are repeated at least 10 times.

26. The separation matrix of claim 1 , wherein the multimer is linked by linkers that comprise or consist essentially of a peptide sequence defined by, or having at least 90% identity, with an amino acid sequence selected from the group consisting of SEQ ID NO: 96-SEQ ID NO: 106.

27. The separation matrix of claim 1 , wherein the multimer is linked by linkers that do not consist of the peptides selected from the group consisting of SEQ ID NO: 107-SEQ ID NO: 110.

28. The separation matrix of claim 16 , wherein the multimer is linked by linkers that comprise or consist essentially of a peptide sequence defined by, or having at least 90% identity, with an amino acid sequence selected from the group consisting of SEQ ID NO: 96-SEQ ID NO: 106.

29. The separation matrix of claim 16 , wherein the multimer is linked by linkers that do not consist of the peptides selected from the group consisting of SEQ ID NO: 107-SEQ ID NO: 110.